Viruses are known to alter populations of microorganisms, such as bacteria, fungi, algae, and protozoa. It has been estimated that, in nature, as many as one-third of all bacteria may be attacked by viruses each day. The destruction of microorganisms by viruses results in fluctuations of microbial populations in the environment, which is referred to as “cycling” of microbial populations. For example, populations of microorganisms increase in concentration until viruses contact and infect susceptible microorganisms, which are referred to as host microorganisms or “hosts.” Viral infections of microorganisms decrease the number of available susceptible host microorganisms, and correspondingly increase the number of viruses. Without hosts to infect, many viruses are eventually destroyed by exposure to natural elements, such as ultraviolet light from the sun and enzymes in the environment. Thus, virus numbers decline, while host microorganism populations consequently increase. Such cycling of microbial populations in nature is common. Although it is somewhat difficult to detect and study viruses that attack microorganisms other than bacteria, those of skill in the art are aware that all populations of microorganisms (e.g., algae, rickettsiae, fungi, mycoplasmas, protozoas) are controlled and cycled in a similar manner by viruses that are capable of infecting and destroying such microorganisms.
Bacterial viruses, which are also referred to as “bacteriophages” or “phages,” are ubiquitous and can be isolated from all bacterial populations where hosts can be cultivated and used for isolation. Phages are naturally occurring entities that are found in or on animals (including humans), plants, soil, and water. Viruses which infect algae, molds, mycoplasmas, protozoa, rickettsiae, yeasts, and other microorganisms are also known.
Two methods are typically employed in order to determine the concentration, which is also referred to as “quantification,” of viruses in natural environments. First, electron microscopy may be used to visualize and count total viral particles in a sample of known size. Second, viable viruses may be cultured, or grown, and counted. An exemplary method of quantification by culturing and counting includes a technique which is typically referred to as a plaque assay. In plaque assays, the viruses that are to be quantified are mixed with a predetermined concentration of host cells and transferred to a liquid (e.g., buffer, mineral salts diluent, or broth). The mixture is then transferred to a semisolid growth medium. The concentration of host cells must be sufficiently great to form a confluent layer, which is typically referred to as a “lawn,” in the semisolid growth medium as the cells grow. During incubation of the phage-host mixture, many of the viable viruses infect host cells. Subsequently, new viruses are produced within infected host cells, which are eventually destroyed, or “lysed,” so that new viruses may be released therefrom. The new viruses then attack and eventually lyse cells that are adjacent to host cells from which the new viruses were released. This spread of infection, which continues as long as host cells are metabolizing, results in formation of clear areas, which are typically referred to as “plaques,” in the host cell lawn. The number of viruses that were present in the original mixture is determined by counting the number of plaques that are formed in the host cell lawn. Accordingly, viruses that are quantified by this method are referred to as plaque-forming units (“PFU”).
In order to quantify all of the various types of viruses in an environmental sample by culturing host cells and counting PFUs, host cells for each of the different viruses in the sample must be cultured. Many types of microorganisms in a given environmental sample are not known. Some of the known microorganisms cannot be cultivated. Therefore, the number of viruses that are present in a given environment may be underestimated when quantified by culturing and counting. Although it is estimated that one gram of soil includes as many as 108 to 109 microorganisms, quantification techniques such as direct plate counting, selective isolation, microscopy, and reassociation kinetics of total DNIA isolated from soil suggest that only a very small percentage of these microorganisms can be cultured. Thus, the development and application of direct electron microscopic counting methods have provided a better understanding of the number of viruses that are present in various environments, as well as the impact that viruses have in reducing microbial populations.
Phages have been quantified in water. Bergh et al. (1989), High abundance of viruses found in aquatic environments, Nature 340:467, used electron microscopy to determine the total concentration of bacterial viruses in a natural, unpolluted Norwegian lake. Phage concentrations of up to about 2.5×108 phages/ml were found in the water. Bacterial counts were as high as about 1.5×107 cells/ml. From these relative concentrations of phage and bacteria, it was estimated that as many as one-third of the bacterial population experiences one or more phage attacks each day. Similarly, Demuth et al. (1993) Direct electron microscopy study on the morphological diversity of bacteriophage populations in Lake Plussee, Appl. Environ. Microbiol. 59:3378, determined that phage levels in a German lake without sewage influences were as high as about 108 phages/ml of lake water. As many as eleven morphologically different phages were identified in the water samples.
Phages have also been quantified in soil. Using the culturing and counting method, with Bacillus stearothermophilus as the host cell, Reanny, D. C. and Marsh S. C. N. (1973). The ecology of viruses attacking Bacillus stearothermophilus in soil Soil. Biol. Biochem. 5:399, reported that, on average, about 4.0×107 PFUs that would infect B. stearothermophilus were present in a gram of soil. Only phages against a single host were, however, quantified in the Reanny and Marsh study. Thus, had other bacterial hosts been tested along with B. stearothermophilus, or had electron microscopy quantification techniques been employed, phage counts would probably have been much higher.
Phages are also present in foods. Kennedy et al. (1986) Distribution of coliphages in various foods. J. Food Protect. 49:944, found Escherichia coli and phages that attack E. coli (“coliphages”), in 11 of 12 tested foods, each of which are available in many retail markets. For example, all ten ground beef samples tested by Kennedy et al. were contaminated with coliphages. Coliphages were also present in samples of fresh chicken, fresh pork, fresh oysters, fresh mushrooms, lettuce, chicken pot pie, biscuit dough, deli loaf deli roasted turkey and packaged roasted chicken. Similarly, Gautier et al. (1995) Occurrence of Propionibacterium freudenreichii bacteriophages in Swiss cheese, Appl. Environ. Microbiol. 61:2572, detected Propionibacterium freudenireichii phage concentrations of about 7×105 PFU/g in Swiss cheese.
Both undesirable and beneficial microorganisms are present in the environment. Viruses infect and destroy both beneficial and undesirable microorganisms. Soil microorganisms that enhance plant growth and microorganisms that degade toxic substances are exemplary of beneficial microorganisms in the environment. Undesirable microorganisms include pathogenic microorganisms and algae that cause algal blooms and fish kills.
In addition to naturally occurring microbial populations, in recent decades disease-causing microorganisms resistant to antibiotics have become epidemic in many hospitals, and have been notoriously difficult to control. During the past fifty or more years, the widespread use of antibiotics has resulted in the selection of antibiotic-resistant bacterial strains. Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Salmonella typhi, Hemophilus ducreyi, Hemophilus influenzae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, various Shigellaspecies, members of the Enterobacteriaceae and Pseudomonas families, and other bacterial species are resistant to many of the conventionally employed antibiotics. Infections that are acquired during hospitalization, which are typically referred to as nosocomial infections, cause an estimated 60,000 deaths per year, and require treatment, which has been estimated to cost about $4.5 billion per year recently.
Statistics from the Centers for Disease Control and Prevention (CDC) indicate that the majority of nosocomial infections are caused by E. coli, S. aureus, coagulase-negative staphylococci, enterococci, pneumococci, and pseudomonads. In addition, according to the 1996 World Health Organization (WHO) annual report, “drug-resistant strains of microbes have evaded common treatments for tuberculosis, cholera, and pneumonia.”
Consequently, the occurrence of infections that are caused by antibiotic-resistant bacteria has steadily increased in hospitals localized communities, and at-risk populations worldwide since the 1940s, shortly after antibiotics were first used for treating bacterial infections. For example, in 1941 practically all strains of S. aureus throughout the world were susceptible to penicillin G. By 1944 however, some strains of S. aureus were capable of making penicillinase, which is also typically referred to as β-lactamase, which degrades penicillin. In 1996, some strains of S. aureus were not only resistant to various forms of penicillin, but also to six of the seven other antibiotics that are conventionally used to treat S. aureus (“staph”) infections.                1) Since 1988, the potential for selection of vancomycin-resistant mutants was a concern in that such resistance had been identified in Gram-positive bacteria, such as vanconmycin-resistant E. faecalis, or faecium (“VREF”); VEEF are also of great concern to health care professionals due to their deadly combination of antibiotic resistance, rapid spread, and high mortality rates in patients with VREF-associated infections.        2) Infections by methicillin-resistant S. aureus (“MRSA”) pose an especially serious public health threat. MRSA typically display various patterns of multiple-drug resistance (i.e., are resistant to multiple types of antibiotics). Many strains of MRSA are susceptible only to the antibiotic vancomycin.        3) Although new and alternative drugs for treating infections of antibiotic-resistant strains of bacteria have been developed and discovered, many bacteria also develop resistance to such new and alternative drugs. For example, certain MRSA strains quickly developed resistance to the antibiotic ciprofloxacin. Moreover, in 1997, a strain of S. aureus was isolated from an infection that resisted 29 days of vancomycin treatment. To put the threat posed by this S. aureus strain in perspective, this S. aureus strain was categorized by the CDC as having intermediate resistance somewhat short of full resistance, and was labeled a medical red alert. It was reported that if MRSA strains which have resistance to vancomycin develop, death rates for all surgeries, including elective surgeries, may increase.        4) In 2001 the isolation of MRSA from three heart patients at McKay-Dee Hospital in Ogden, Utah, resulted in closure of its cardiac surgical units to all but emergency surgeries. Subsequently, vancomycin-resistant S. aureus (VRSA) have been isolated from clinical patients in Michigan (2002), Pennsylvania (2003) and New York (2004).        
Similarly, about half of the known strains of S. pneumoniae are resistant to penicillins, which have conventionally been employed as the initial and primary treatment for S. pneumoniae infections. Some S. pneumoniae strains are resistant to cephalosporin antibiotics, which have conventionally been employed as a secondary treatment for S. pneumoniae infections. Penicillin and cephalosporin-resistant S. pneumoniae strains may be treated with vancomycin. The use of vancomycin, however, is undesirable because of severe side effects that vancomycin has on many patients and the possibility that vancomycin-resistant strains of S. pneumoniae may emerge.
The problem of antibiotic resistance is further compounded by the fact that microorganisms may transfer genetic information, which is referred to as “genes” or “DNA” for simplicity. Methods by which microorganisms, such as bacteria, can transfer DNA, and even entire genes, include conjugation, transformation, and transduction. Various genes, including genes that impart bacteria with resistance to antibiotic drugs, may be transferred from a first, or donor, microorganism to a second, or recipient, microorganism. In addition to transferring genes for antibiotic resistance, microorganisms may transfer genes that enable a microorganism to produce toxins, which are typically harmful to an infected host. Virulence factors, which determine the types of hosts and host cells that a microorganism can infect may also be transferred from one microorganism to another.
In conjugation, plasmid or chromosomal DNA is transferred directly from a donor microorganism to a recipient microorganism by means of specialized pili or “sex pili,” which are small, hollow, filamentous appendages, which bind to and penetrate the cell membranes of recipient microorganisms. Conjugation is a process by which genes that code for antibiotic resistance in the “donor” microorganism pass to a recipient microorganism, transforming the recipient into an antibiotic-resistant microorganism.
Transformation is the transfer of DNA that has been released into the environment by a donor microorganism and incorporated by a recipient microorganism. Transformation experiments have been conducted in sterile soil that was inoculated with two parental strains of Bacillus subtilis with differentially marked, or tagged, DNA. Bacteria were isolated which carried the markers of both parental strains. Even under the best laboratory conditions, however, transformation is relatively inefficient and requires high densities of donor DNA and recipient cells. Conditions that would permit transformation in many microorganisms are typically not present in a natural, or uncontrolled, environment. Consequently, transformation is typically perceived as a laboratory phenomenon.
Transduction is the transfer of host genes to recipient microorganisms by viruses, such as phages. There are two kinds of phages, virulent, or lytic, and temperate. When a host cell is infected with a virulent phage, new phages, which are typically referred to as progeny, are grown in the host cell, and the host cell is subsequently lysed, or destroyed, so that the progeny may be released. In contrast, temperate phages typically infect host cells without destroying their host. Following infection of a host cell, temperate phages typically incorporate their genetic information into the DNA of the host cell. Many temperate phage-infected host cells can be subsequently induced, by ultraviolet light, mutagens, or otherwise, to enter a lytic cycle, wherein genetic information of the temperate phage produces progeny which then lyse the host cell.
Transduction of host DNA may be either “specialized” or “generalized.” In specialized transduction, a temperate phage's genome is integrated into the chromosome of a host donor microorganism without lysing the host. The phage genome that was inserted into the host chromosome is referred to as a “provirus,” or “prophage,” and is passively replicated as the host cell and its chromosome replicate. Bacteria that carry proviruses are said to be lysogenic. Certain events, such as exposing the host microorganism and the provirus to ultraviolet light, may cause the provirus to act as a virulent phage, whereby the provirus is excised from the bacterial chromosome. Such excised proviruses may carry bacterial genes, or “donor” genes, with them. Upon infecting a new host, or recipient microorganism, these “donor” genes may be expressed, which may alter the phenotype, or physical gene expression, of the recipient microorganism.
Temperate and, possibly, some virulent phages may affect generalized transduction. During viral replication, a section of DNA of the donor microorganism, which is referred to as a “donor” gene, rather than the phage genome, may be enclosed inside a phage head. Phages that include only DNA of a host microorganism are referred to as transducing particles. A typical phage is only capable, however, of containing about one percent of the chromosome of a host, or “donor,” microorganism. Thus, the simultaneous transfer of more than one gene by a single transducing particle is unlikely. Since transducing particles do not include a phage genome, transducing particles cannot produce progeny upon infecting a recipient microorganism. Instead, the donor gene has to be incorporated into the chromosome of the recipient microorganism. If the recipient microorganism is infected with only one transducing particle, it will survive and its phenotype may be altered by the integrated donor gene. It is very important to remember if the multiplicity of infection (“MOI”) of transducing particles per recipient microorganism is high, the cell will probably be destroyed, which is typically referred to as “lysis from without.”
The transfer of genetic information from one microorganism to another may have beneficial or undesirable effects. For example, a beneficial transfer of genetic information was disclosed by Chakrabarty, A. M. (1996) Microbial degradation of toxic chemicals; Evolutionary insights and practical considerations, ASM News 62:130. Microorganism-rich soil was introduced into a chemostat which contained a single industrial pollutant as a nutrient. In less than a year, pseudomonads which had acquired all of the enzymes needed to degrade the pollutant were isolated from the soil.
Similarly, genes that exhibit undesirable traits may also be transferred. Examples of such detrimental gene transfer include transfer of genes carrying resistance to antibiotics, and genes that code for production of toxins, such as shiga, diphtheria, and botulism toxins. Outbreaks of toxin-related diseases, such as toxic shock syndrome in 1980, the “flesh-eating streptococci” of 1994, and illnesses caused by E. coli 0157:H7 in undercooked hamburger, have been traced to the transfer of toxin genes by temperate phages. Genes that code for cholera toxin are also reported to have been transmitted by a temperate phage, which created yet another epidemic strain, Vibrio cholerae 0139.
Viruses have been isolated and employed in treating various types of bacterial infections. U.S. Pat. No. 4,375,734, which issued to Kozloff et al. on Mar. 8, 1983 (“Kozloff”), discloses use of a wild-type phage, Erh1, for protecting plants against frost injury caused by an ice nucleation-promoting bacterium, Erwinia herbicola. The treatment of corn plants with Erh 1 reduced the incidence of ice nucleation damage by about 20% to 25%. Kozloff et al. also discloses that Erh1 killed only about 90% of cultured E. herbicola, which suggests that some of the remaining 10% were resistant to wild-type Erh1.
U.S. Pat. No. 4,828,999, which issued to Jackson, one of the present inventors, on May 9, 1989 (“Jackson”), discloses host range, or “h-mutant,” phages which attack phage-resistant strains of various plant bacteria, and methods of treating bacterially infected plants. The h-mutant phages, compositions containing such phages, and methods of treatment that are disclosed in Jackson are, however, limited to phages for plant bacteria and the treatment of plants infected with such bacteria.
Similarly, some measures have been taken to address the problem of bacterial diseases in humans, and to otherwise control and prevent bacterial growth. patent application Ser. No. 08/222,956 (the '“956 application”), which was published on Oct. 12, 1995 as WO 95/27043, discloses a type of phage therapy whereby mutant phage strains are introduced into a bacterially infected host. The mutant phages, which are thought to be resistant to degradation by the bacterially infected host's defense systems, particularly organs of the reticulo endothelial system, are believed to attack the harmful bacteria with which the host is infected. Thus, phages of the '956 application are believed to act as an in vivo antibacterial agent, and may be used either alone or as an adjunct to antibiotic therapy.
Although phages disclosed in the '956 application are introduced into bacterially infected hosts for the purpose of attacking undesirable bacteria, these phages included not only lytic, but also temperate viruses which are able to transfer pieces of donor bacterial DNA to recipient bacteria. Further, the '956 application lacks any disclosure that phages disclosed therein are able to attack, and thereby prevent or otherwise control the further growth of, phage-resistant bacterial strains.
Shortly after the discovery of phages as lytic agents of bacteria by Twort in 1915 and by d'Herelle in 1917, the investigation of their use for treating bacterial infections, which is typically referred to as phage therapy, began. Various phages are active against bacteria of many diseases in plants and animals, such as mammals. Phages that are active against bacteria which cause human diseases, such as anthrax, bronchitis, diarrhea, scarlet fever, typhus, cholera, diphtheria, gonorrhea, paratyphus, bubonic plague osteomyclitis, and other bacterially induced diseases, are known. While many in the art were initially convinced of the efficacy of phage therapy, particularly in controlling cholera, many phages were ineffective for in vivo treatment. It was believed that such ineffectiveness was due to the inactivation of phage by the host's immune system when administered parenterally, denaturation by gastric juices when taken orally, and the rapid emergence of phage-resistant bacterial mutants.
With the introduction and use of antibiotics, and their initial effectiveness in controlling bacterial diseases, much of the research for using phages as therapeutic agents ceased. Recently, phage therapy was successfully employed to treat nosocomial infections caused by antibiotic-resistant bacteria and certain opportunistic pathogens, namely, pyogenic infections and septicemias, especially staphylococcal, but also pseudomonads, enterobacteria (E. coli, Klebsiella, Protius, Providencia, Serratia), injuries (infected wounds and burns, postoperative infections, osteomyelitis), diseases of the skin and subcutaneous tissue (furunculosis, abscesses, acute lymphangitis, decubitus ulcers), urinary infections (chronic cystitis and pyelonephritis), respiratory diseases (sinusitis, mucopurulent bronchitis, pleuritis) and other diseases, for example, infantile diarrhea caused by enteropathogenic E. coli (7,8). In treating bacterial infections, phages may be administered orally in liquids, tablets and capsules, topically by aerosols and direct application, and intravenously. Phage therapy was conducted alone and in combination with antibiotics. Phages were also used as antiseptics, including uses such as disinfecting operating rooms, surgical instruments and lesions on patients, and medical care professionals.
Microorganisms such as bacteria can develop phage-resistant strains, however. Thus, phage therapy (or virus therapy for non-bacterial microorganisms) is somewhat undesirable from the standpoint that virus-resistant strains of a target strain of microorganism may persist in an infected host that is being treated, or in any other treated environment.
Conversely, many beneficial microorganism populations are threatened by viruses that will interfere with the beneficial properties of such microorganisms. Exemplary beneficial processes that are facilitated by microorganisms include industrial fermentation (e.g., in making food products), bioremediation of toxic chemicals, pollutants, and other undesirable substances, leaching of metals from low grade ores, extraction of petroleum and related products from shale, and drug manufacture. The efficiency of many beneficial processes is degraded by the ubiquitous nature of many viruses that will attack the microorganisms that facilitate these processes.
Thus, a need exists for an alternative method of controlling, reducing, or eliminating microorganism populations, which method addresses the ever-increasing emergence of antimicrobial resistance and the virus-resistance of microorganisms. A need also exists for a treatment which selects and destroys undesirable microorganisms while permitting beneficial microorganisms to survive. A need also exists for providing virus-resistant beneficial microorganisms.